ProjectSummary Aggressivemetastaticbreastcancerisresponsibleforthedeathsofmorethan40,000peopleperyearinthe U.S.,despitethebesteffortsofresearchersandclinicaloncologists.Ihaverecentlydiscoveredapotentially importantmechanismregardingthegenerationofauniquesecretomebybreastcancercells,whichmakes an essential contribution to their invasiveness and metastatic capability. Specifically, I have delineated an intriguing connection between the down-regulation of Sirtuin 1 (Sirt1), a NAD-dependent deacetylase, in aggressive breast cancer cellsandthecorresponding reduction in the expression ofa major subunit ofthe vacuolarATPase(V-ATPase),whichresultsintheimpairmentoftheirlysosomesandconsequently,dramatic changes in their secretome. These changes include a significant increase in the number of exosomes generatedbybreastcancercells,andanenrichmentintheirubiquitylatedproteincargo.Exosomesaresmall (extracellular) vesicles, ~30-150 nm in size, that contain a wide range of cargo including proteins, RNA transcripts,microRNA,andevenDNA.Theyfunctionasmediatorsofintercellularcommunicationandhave beenimplicatedinanumberofaspectsofcancerprogression,includingthepromotionofchemo-resistance andtheformationofapre-metastaticniche.Becauseexosomesarealsoattractivevehiclesforthedelivery of therapeutic agents, studies aimed at determining how exosomes are formed and released, as well as characterizingtheirfunctionalproperties,arebeingextensivelypursued.Thus,thesefindingsnowhighlight how aggressive breast cancer cells generate exosomes containing unique cargo, which contribute to the metastatic capability of breast cancer cells. I further discovered that the down-regulation of Sirt1 in breast cancercellsresultsinasignificantincreaseinthesecretionofsolublehydrolases,inparticular,cathepsins. Collectively, these components making up the secretome of aggressive breast cancer cells give rise to a markedenhancementinmigratoryandinvasiveactivity.IntheF99phaseofthisapplication,thesediscoveries will be extended by determining the underlying mechanisms by which the down-regulation of Sirt1 in aggressivecancercellsleadstoareducedexpressionoftheV-ATPase(Aim1).Aparticularemphasiswill be to identify the Sirt1 substrate that is directly responsible for regulating the stability of the RNA transcript encoding one of the major subunits of the V-ATPase. In the K00 phase of the proposal (Aim 2), a research/trainingenvironmentwillbesoughttodevelopanimalmodelsthatwillfurtherestablishthefunctional connectionbetweenSirt1,thev-ATPaseandlysosomalfunction,anddemonstratehowthiscontributestothe metastaticstate.Theultimategoalofmystudieswillbetohighlightstrategiesthataltertheseconnectionsin amannerthatleadstonewanti-cancertherapies.